Abstract: In this experiment, the inorganic selenium conversion rate of Hansenula polymorpha (H.polymorpha) and the yield of intracellular glutathione (GSH) were used as evaluation indexes.The effects of parameters such as sodium selenite (Na₂ SeO₃) addition, addition time, inoculum size were studied by single factor experiment and orthogonal experiment to determine the optimal fermentation process for preparing selenium-glutathione-enriched H.polymorpha.The physiological function of selenium -glutathione-enriched H.polymorpha in rats was analyzed by MTT colorimetric assay. The optimal condition of inorganic selenium produced by H.polymorpha was following:inoculation amount of 4%, Na₂ SeO₃ with a final concentration of 30 μg/mL addition to the fermentation medium after 12 h cultivation, the fermentation period of 48 h, the culture temperature of 37℃ and the shaking speed of 120 r/min. Under this condition Na₂ SeO₃ conversion rate and intracellular GSH yield of H.polymorpha reached 97.28% ±1.89% and (215.21 ±3.01) mg/L, respectively.Meanwhile, the serum and liver GSH levels of SD rats in the selenium-glutathione-enriched H.polymorpha group were increased by 39.5% (p < 0.01) and 45.2% (p < 0.001), T-AOC with the increase of 46.3% (p < 0.01), 45.2% (p < 0.05), GSH-Px with the increase of 48.3% (p < 0.001), 52.3% (p < 0.01), SOD with the increase of 68.4% (p < 0.01), 45.1% (p < 0.05), CAT with the increase of 39.5% (p < 0.01), 49.6% (p < 0.001), and MDA with the decrease of 21.3% (p < 0.05), 38.5% (p < 0.01), respectively, which indicated that selenium-glutathione-enriched H.polymorpha could significantly increase the antioxidant activity of rats.The results would provide novelty pathways for preparing inorganic selenium and glutathione by H.polymorpha and offered a technical support for its application in food development.